The present invention relates generally to the art of separating charged molecular species, and, in particular, to separation media that are used for capillary electrophoresis.
Gel electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Charged molecular species such as proteins, peptides, nucleic acids and oligonucleotides are separated by causing the species to migrate in a buffer medium under the influence of an applied electric field. The buffer medium normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent to promote the separation and to minimize the occurrence of mixing the species being separated.
Until recently, electrophoretic separations were conducted in gel slabs or open gel beds that were typically fabricated of agarose or cross-linked polyacrylamide materials. More recently, capillary electrophoresis (“CE”) using a polymer gel or solution as a separation medium has been used for the separation of charged molecules, such as DNA. Capillary electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of charged molecules. Furthermore, capillary electrophoresis can provide quantitative information about a sample using very small amounts of the sample, gel (or polymer solution) and buffer relative to traditional slab gel processes. Moreover, high-resolution separation of charged macromolecules having different effective charges has been achieved.
Typically, the capillary columns used in capillary electrophoresis are fabricated from fused silica tubing having diameters on the order of 25 μm to 200 μm and lengths from about 30 cm to about 200 cm. The column interior is filled with a gel or solution separation medium, along with a buffer. Electrophoretic techniques are used to separate charged molecular species.
The separation medium is one of the most important parameters in CE since it determines the migration behavior, including the resolution, of charged molecular species (e.g., DNA fragments). Polymer solutions are widely used at present for such media.
Several synthetic hydrophilic homopolymers have been developed and tested as DNA separation media, including linear polyacrylamide (LPA); and alkyl-substituted derivatives, such as poly-N,N-dimethylacrylamide (PDMA), poly-N-acryloylamino-ethoxyethanol (PAAEE), polyacryloylaminopropanol (PAAP), poly(acryloylaminoethoxy)ethylglucopyranoside (PAEG), polyethylene glycol (PEG), and polyethylene oxide (PEO).
The electrophoresis community largely agrees that highly entangled solutions of LPA have generally provided the best sequencing separations, making it possible to separate DNA molecules differing by a single base for fragments up to 1300 bases in length. (Zhou et al., Anal. Chem. 72:1045–52 (2000).) The excellent performance of LPA is believed to result in part from its hydrophilic nature.
Although LPA provides superb DNA separations for long read sequencing, it has the disadvantage of requiring the use of a stable capillary wall coating for suppression of electro-osmotic flow (EOF) and prevention of analyte absorption. Electro-osmotic flow is caused by the inability of a separation medium to bind directly to the inner wall of the capillary tubes. This highly problematic flow occurs upon the application of an electric field during separations. An electrical double layer is generated by the attraction of soluble buffer ions to the charged surface of the capillary wall. An excess in the local concentration of ions in the solution near the wall develops so that charge neutrality may be maintained proximal to the wall. The net result is that charged channel walls engender a bulk flow of fluid during electrophoresis. Electro-osmotic flow results in highly unsatisfactory separation results.
Traditional methods aimed at preventing EOF include introducing a compound that binds to the inner surface of a capillary tube wall prior to injecting the separation medium into the tube. For example, U.S. Pat. No. 5,447,617 to Shieh describes covalently bonding polybutadiene to the inner surface of a capillary tube, introducing acrylamide monomers therein and copolymerizing the acrylamide with the polybutadiene. Such precoating techniques increase cost and give rise to problems such as capillary fouling, coating inhomogeneity, and limited shelf life for coated capillary tubings.
To avoid the problem of EOF, less hydrophilic polymers have been used as separation media. For example, poly(vinyl pyrrolidone) (PVP) has been used as a DNA sequencing matrix. Since PVP dynamically coats the capillary tube walls, the need for precoating the walls is obviated. However, the separation results of PVP are much poorer than those achieved with LPA. The separation of only slightly more than 300 contiguous DNA bases has been achieved with PVP. The limiting characteristic of PVP is its excessive nonspecific (hydrophobic) interaction between the sieving matrix and fluorescent dyes used during separations. Such interaction obscures the separation of larger DNA fragments.
The development of a high performance separation medium, which possesses high sieving ability, low viscosity and dynamic coating ability, will facilitate the automation of CE and further enhance its performance. However, homopolymers which posses all these properties have not heretofore been found. Accordingly, mixtures of different homopolymers have been investigated.
Mixtures of the same polymer, such as PEO, hexaethyl cellulose (HEC) and LPA, with different molecular weights and mixtures of two modified polysaccharides, i.e., agarose and HEC, have been found to produce a better resolution for both small and large DNA fragments. However, a mixture of two polymers with totally different chemical structures has never been successfully used. Kim et al. (Kim, Y., Yeung, E. S., J. Chromatogr. A., 1997, 781, 315–325) tried to use a mixture of PEO and hexapropyl cellulose (HPC) for DNA sequencing and found the separation to be very poor. The failure was attributed to the incompatibility of the two polymers.
Another challenge in CE is the introduction of separation matrices, which are usually quite viscous, into the narrow capillary tubes. The polymerization of polyacrylamide within a capillary tube avoids the problem of forcing the polymeric solution into the capillary tube or microchannels. It is also desirable to remove “used” polymer matrix out of the capillary tube after each use and refill the capillary tube with fresh matrix. However, once the polyacrylamide is polymerized within a capillary tube, the polymerized gel cannot be easily removed from the capillary tube.
Recently, copolymers with viscosity dependent behavior have been employed. Such copolymers allow the loading and unloading of the medium in low viscosity states, and electrophoretic separation in a high viscosity state. For example, matrices based on a hydrophilic backbone of LPA with short grafts of low molar mass (N-isopropylacrylamide) have been described. (Sudor et al. Electrophoresis 22:720–8 (2001).) Also, matrices based on a copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (“P(DMA/DEA)”) has been tested. (Buchholz et al. Anal Chem 73:157–64. (2001).) However, the synthesis of copolymers is difficult to control and reproduce, leading to unreliable results.
A successful separation medium for electrophoresis would include properties such as stability; neutrality; appropriate mesh size; dynamic coating ability (to efficiently suppress electro-osmosis); low viscosity; the use of a minimal amount of medium material; and providing good separation results and long read lengths. Before the present invention, a medium that would provide some desirable properties, e.g., a long read length, typically would not allow for other desirable properties, e.g., dynamic coating ability.